Malignant insulinoma is a critical cancer form with a poor prognosis. Because cure by surgery is infrequent, effective chemotherapy is in demand. Induction of cell death in tumor cells by proteasome inhibitors is emerging as a potential strategy in cancer therapy. Here we investigated whether inhibition of the proteasome has an antitumorigenic potential in insulinoma cells. Exposure of mouse betaTC3 insulinoma cells to the proteasome inhibitor N-Acetyl-Leu-Leu-Nle-CHO (ALLN) reduced cell viability, activated caspase-3, induced apoptosis, and suppressed insulin release. Treatment with ALLN also resulted in phosphorylation of c-jun N-terminal kinase (JNK) and an increase in in vitro phosphorylation of c-jun. In insulinoma cells with impaired JNK signaling, ALLN-induced apoptosis was significantly suppressed. Another proteasome inhibitor, lactacystin, also stimulated JNK activation, caused activation of caspase-3, suppressed cell viability, and induced apoptosis in betaTC3 and rat INS-1E cells. Both ALLN and lactacystin caused a marked decrease in the cellular amount of the JNK scaffold protein JNK-interacting protein 1/islet-brain-1. In primary pancreatic rat islet cells, proteasome inhibition reduced insulin secretion but had no impact on cell viability and even partially protected against the toxic effect of proinflammatory cytokines. Our findings demonstrate that proteasome inhibitors possess antitumorigenic and antiinsulinogenic effects on insulinoma cells.